Bone Tissue Regeneration by Collagen Scaffolds with Different Calcium Phosphate Coatings: Amorphous Calcium Phosphate and Low-Crystalline Apatite

Surface-mineralized collagen sponges have attracted much attention as scaffolds for bone tissue engineering. Recently, we developed amorphous calcium phosphate (ACP) and low-crystalline apatite coating processes on collagen sponges. In the present study, we applied these coating processes to granular collagen sponges (referred to as Col) to compare the bone tissue regeneration capabilities of ACP-coated and apatite-coated Col (referred to as Col-ACP and Col-Ap, respectively) using a rat cranial bone defect model. According to micro-CT and histological analyses, Col-Ap enhanced bone tissue regeneration compared to Col, whereas Col-ACP did not. These results not only demonstrated the superior bone tissue regeneration capability of Col-Ap, but also indicated limitations of the in vitro simulated body fluid (SBF) test used in our previous study. Despite the apatite-forming ability of Col-ACP in SBF, it was ineffective in improving bone tissue regeneration in vivo, unlike Col-Ap, most likely due to the quick resorption of the ACP coating in the defect site. The present results clarified the importance of the coating stability in vivo and revealed that the low-crystalline apatite coating was more beneficial than the ACP coating in the fabrication of surface-mineralized collagen sponges for use as bone tissue engineering scaffolds.

Silicate/zinc-substituted strontium apatite coating improves the osteoinductive properties of β-tricalcium phosphate bone graft substitute

Background β-Tricalcium phosphate (β-TCP) is a popular synthetic bone graft substitute with excellent osteoconductive properties and bioabsorbability. However, its osteoinductive properties are inferior to those of autologous or allogeneic bone. Trace elements such as strontium (Sr), silica (Si), and zinc (Zn) have been reported to promote osteogenesis in materials. In this study, we aimed to determine whether a Si/Zn-substituted Sr apatite coating of β-TCP could enhance osteoinductive properties. Methods The apatite-coated β-TCP disks were prepared using nanoparticle suspensions of silicate-substituted Sr apatite (SrSiP) or silicate- and Zn-co-substituted Sr apatite (SrZnSiP). Bone marrow mesenchymal cells (BMSCs) from rat femur were cultured and subsequently seeded at a density of 1.0 × 106/cm2 onto apatite-coated and non-coated β-TCP disks. In vitro, the β-TCP disks were then placed in osteogenic medium, and lactate dehydrogenase (LDH) activity was measured from supernatants after culture for 2 days. Additionally, after culture for 14 days, the mRNA expression of genes encoding osteocalcin (OC), alkaline phosphatase (ALP), bone morphogenetic protein-2 (BMP-2), and vascular endothelial growth factor (VEGF) was evaluated by qRT-PCR. In vivo, the β-TCP disks were transplanted subcutaneously into rats that were sacrificed after 4 weeks. Then, the harvested disks were evaluated biochemically (ALP activity, OC content, mRNA expression of OC, ALP, BMP-2, and VEGF measured by qRT-PCR), radiologically, and histologically. Results Significantly higher mRNA expression of almost all evaluated osteogenic and angiogenic genes was observed in the SrZnSiP and SrSiP groups than in the non-coated group, with no significant cytotoxicity elicited by the apatite coating in vitro. Moreover, in vivo, the SrZnSiP and SrSiP groups showed significantly higher osteogenic and angiogenic gene expression and higher ALP activity and OC content than the non-coated group (P < 0.05). Radiological and histopathological findings revealed abundant bone formation in the apatite-coated group. Conclusions Our findings indicate that apatite coating of β-TCP improves osteoinductive properties without inducing significant cytotoxicity.

Silicate/zinc-substituted strontium apatite coating improves the osteoinductive properties of β-tricalcium phosphate bone graft substitute

Background: β-Tricalcium phosphate (β-TCP) is a popular synthetic bone graft substitute with excellent osteoconductive properties and bioabsorbability. However, its osteoinductive properties are inferior to those of autologous or allogeneic bone. Trace elements such as strontium (Sr), silica (Si), and zinc (Zn) have been reported to promote osteogenesis in materials. In this study, we aimed to determine whether a Si/Zn-substituted Sr apatite coating of β-TCP could enhance osteoinductive properties.Methods: The apatite-coated β-TCP disks were prepared using nanoparticle suspensions of silicate-substituted Sr apatite (SrSiP) or silicate- and Zn-co-substituted Sr apatite (SrZnSiP).Bone marrow mesenchymal cells (BMSCs) from rat femur were cultured and subsequently seeded at a density of 1.0 × 106/cm2 onto apatite-coated and non-coated β-TCP disks.In vitro, the β-TCP disks were then placed in osteogenic medium, and lactate dehydrogenase (LDH) activity was measured from supernatants after culture for 2 days. Additionally, after culture for 14 days, the mRNA expression of genes encoding osteocalcin (OC), alkaline phosphatase (ALP), bone morphogenetic protein-2 (BMP-2), and vascular endothelial growth factor (VEGF) was evaluated by qRT-PCR. In vivo, the β-TCP disks were transplanted subcutaneously into rats that were sacrificed after 4 weeks. Then, the harvested disks were evaluated biochemically (ALP activity, OC content, mRNA expression of OC, ALP, BMP-2, and VEGF measured by qRT-PCR), radiologically, and histologically.Results: Significantly higher mRNA expression of almost all evaluated osteogenic and angiogenic genes was observed in the SrZnSiP and SrSiP groups than in the non-coated group, with no significant cytotoxicity elicited by the apatite coating in vitro. Moreover, in vivo, the SrZnSiP and SrSiP groups showed significantly higher osteogenic and angiogenic gene expression and higher ALP activity and OC content than the non-coated group (P < 0.05). Radiological and histopathological findings revealed abundant bone formation in the apatite-coated group.Conclusions: Our findings indicate that apatite coating of β-TCP improves osteoinductive properties without inducing significant cytotoxicity.

The Use of Simulated Body Fluid (SBF) for Assessing Materials Bioactivity in the Context of Tissue Engineering: Review and Challenges

Some special implantable materials are defined as “bioactive” if they can bond to living bone, forming a tight and chemically-stable interface. This property, which is inherent to some glass compositions, or can be induced by applying appropriate surface treatments on otherwise bio-inert metals, can be evaluated in vitro by immersion studies in simulated body fluid (SBF), mimicking the composition of human plasma. As a result, apatite coating may form on the material surface, and the presence of this bone-like “biomimetic skin” is considered predictive of bone-bonding ability in vivo. This review article summarizes the story and evolution of in vitro bioactivity testing methods using SBF, highlighting the influence of testing parameters (e.g., formulation and circulation of the solution) and material-related parameters (e.g., composition, geometry, texture). Suggestions for future methodological refinements are also provided at the end of the paper.